Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos

The CRISPR-Cas RNA-guided system has versatile uses in many organisms and allows modification of multiple target sites simultaneously. Generating novel genetically modified mouse and rat models is one valuable application of this system. Through the injection of Cas9 protein instead of mRNA into embryos, we observed fewer off-target effects of Cas9 and increased point mutation knock-in efficiency. Large genomic DNA fragment (up to 95 kb) deletion mice were generated for in vivo study of lncRNAs and gene clusters. Site-specific insertion of a 2.7 kb CreERT2 cassette into the mouse Nfatc1 locus allowed labeling and tracing of hair follicle stem cells. In addition, we combined the Cre-Loxp system with a gene-trap strategy to insert a GFP reporter in the reverse orientation into the rat Lgr5 locus, which was later inverted by Cre-mediated recombination, yielding a conditional knockout/reporter strategy suitable for mosaic mutation analysis

Hypoxia destroys the microstructure of microtubules and causes dysfunction of endothelial cells via the PI3K/Stathmin1 pathway

Abstract Background Endothelial cells (EC) are sensitive to changes in the microenvironment, including hypoxia and ischemia. Disruption of the microtubular network has been reported in cases of ischemia. However, the signaling pathways involved in hypoxia-induced microtubular disruption are unknown. The purpose of this study was to investigate the molecular mechanisms involved in hypoxia-induced microtubular disassembly in human umbilical vein endothelial cells (HUVECs). Results HUVECs were cultured under normoxic or hypoxic conditions and pretreated with or without colchicine or paclitaxel. The MTT assay, Transwell assay, trans-endothelial permeability assay, and 5-bromo-2′-deoxy-uridine staining were used to test the survival rate, migration, permeability, and proliferation of cells, respectively. Transmission electron microscopy and phalloidin staining were used to observe the microstructure and polymerization of microtubules. The results show that the functions of HUVECs and the microtubular structure were destroyed by hypoxia, but were protected by paclitaxel and a reactive oxygen species (ROS) inhibitor. We further used western blot, a luciferase assay, and co-immunoprecipitation to describe a non-transcription-independent mechanism for PI3K activation-inhibited microtubular stability mediated by Stathmin1, a PI3K interactor that functions in microtubule depolymerization. Finally, we determined that hypoxia and ROS blocked the interaction between PI3K and Stathmin1 to activate disassembly of microtubules. Conclusion Thus, our data demonstrate that hypoxia induced the production of ROS and damaged EC function by destroying the microtubular structure through the PI3K/stathmin1 pathway

Lactiplantibacillus plantarum NKK20 Increases Intestinal Butyrate Production and Inhibits Type 2 Diabetic Kidney Injury through PI3K/Akt Pathway

Nephropathy injury is a prevalent complication observed in individuals with diabetes, serving as a prominent contributor to end-stage renal disease, and the advanced glycation products (AGEs) are important factors that induce kidney injury in patients with diabetes. Addressing this condition remains a challenging aspect in clinical practice. The aim of this study was to explore the effects of Lactiplantibacillus plantarum NKK20 strain (NKK20) which protects against diabetic kidney disease (DKD) based on animal and cell models. The results showed that the NKK20 can significantly reduce renal inflammatory response, serum oxidative stress response, and AGE concentration in diabetic mice. After treatment with NKK20, the kidney damage of diabetic mice was significantly improved, and more importantly, the concentration of butyrate, a specific anti-inflammatory metabolite of intestinal flora in the stool of diabetic mice, was significantly increased. In addition, nontargeted metabolomics analysis showed a significant difference between the metabolites in the mouse serum contents of the NKK20 administration group and those in the nephropathy injury group, in which a total of 24 different metabolites that were significantly affected by NKK20 were observed, and these metabolites were mainly involved in glycerophospholipid metabolism and arachidonic acid metabolism. Also, the administration of butyrate to human kidney- (HK-) 2 cells that were stimulated by AGEs resulted in a significant upregulation of ZO-1, Occludin, and E-cadherin gene expressions and downregulation of α-SMA gene expression. This means that butyrate can maintain the tight junction structure of HK-2 cells and inhibit fibrosis. Butyrate also significantly inhibited the activation of PI3K/Akt pathway. These results indicate that NKK20 can treat kidney injury in diabetic mice by reducing blood glucose and AGE concentration and increasing butyrate production in the intestine. By inhibiting PI3K pathway activation in HK-2 cells, butyrate maintains a tight junction structure of renal tubule epithelial cells and inhibits renal tissue fibrosis. These results suggest that NKK20 is helpful to prevent and treat the occurrence and aggravation of diabetic kidney injury

Table_1_Reference genes identification for qRT-PCR normalization of gene expression analysis in Cucumis sativus under Meloidogyne incognita infection and Pseudomonas treatment.docx

qRT-PCR is a common and key technical means to study gene expression in biological research. However, reliability and accuracy of quantification by qRT-PCR is entirely dependent on the identification of appropriate reference genes. Cucumber as an economical vegetable is widely cultivated worldwide and is subject to serious nematode infection, especially from M. incognita. Plant could employ beneficial soil bacteria in the rhizosphere to enhance plant adaptability to various stresses. In this study, the optimal reference genes in cucumber under M. incognita stress and Pseudomonas treatment were calculated and confirmed. A total of thirteen candidate reference genes were identified across three different treatments. Of these, geNorm, NormFinder and BestKeeper programs combined RefFinder software identified EF1 and UBI are the most suitable reference gene in the root knot and whole root of cucumber infected M. incognita, respectively, and CACS is the most suitable reference gene in the whole root of cucumber treated by Pseudomonas. The work first validated the most suitable reference genes for the normalization gene expression in cucumber by nematode infected or Pseudomonas inoculated, and these results would facilitate the further research on M. incognita or Pseudomonas soil rhizosphere microbe interaction with cucumber.</p